Diagnosis of Shwachman-Diamond Syndrome

ABSTRACT

The SBDS gene has been identified as the site of mutations associated with SDS. Methods are provided for determining whether a subject is suffering from SDS.

FIELD OF THE INVENTION

The invention relates to methods for diagnosing and treating individualswith Shwachman-Diamond Syndrome and for detecting Shwachman-Diamonddisease carriers. More specifically, the invention relates to theidentification of the Shwachman-Bodian-Diamond Syndrome (SBDS) gene andthe identification of mutations of this gene which are associated withShwachman-Diamond Syndrome.

BACKGROUND OF THE INVENTION

Shwachman-Diamond Syndrome (SDS [MIM 260400]) is an autosomal recessivedisorder with clinical features including exocrine pancreaticinsufficiency, haematological dysfunction, and skeletalabnormalities^(1,2,3). Patients with SDS have a high risk of bone marrowfailure and are at risk of developing acute myelogenous leukaemia (AML).SDS is the second most common cause of pancreatic insufficiency aftercystic fibrosis and involves the failure of development of the exocrinepancreas. Other manifestations include skeletal abnormalities and liverfunction abnormalities, the latter being notable in young patients.

Many SDS patients present with malabsorption and steatorrhea related totheir pancreatic insufficiency. Many such children fail to thrive due tothe malabsorption and also due to their disinclination to eat normallybecause of gastrointestinal upsets. The haematological dysfunction mostconsistently involves neutropenia but can also present asthrombocytopenia or pancytopenia. Serious consequences for SDS patientsinclude recurring severe infections that can be life threatening if thediagnosis is not made with the provision of prompt treatments. Further,traditional methods for treatment of bone marrow failure are generallynot successful in SDS patients at this time but the surveillance andmonitoring of the bone marrow to determine the occurrence ofmyelodysplasia, aplastic anaemia and/or the development of AML doprovide some options for intervention.

It is therefore important for the optimum development and overall longterm prognosis of these children that they are diagnosed as having SDSas early as possible so that infections may be treated with appropriateinterventions, so that blood and bone marrow can be monitored forcellularity (numbers and cell types) and so that pancreatic enzymesupplementation may be instituted to provide adequate or near normalfood absorption.

There are other diseases associated with exocrine pancreaticdysfunction, such as Cystic Fibrosis and Pearson Marrow Syndrome, andother diseases such as congenital neutropenia, Blackfan-Diamond Syndromeand Fanconi Anaemia can mimic the haematological manifestations of SDS.It is important, for proper treatment, that SDS is diagnosed as early aspossible but at present SDS can only be distinguished from otherdiseases causing similar symptoms by complex, symptom-based tests whichmay have to be repeated many times before a conclusion is reached(Rothbaum et al., (2002), J. Pediatrics, v. 141, pp. 266-270; Ginzberget al., (2000), Am. J. Hum. Genet., v. 66, pp. 1413-1416).

There is therefore a real need for a convenient and definitive test,such as a genetic test or a gene product-based immunological test, todiagnose SDS. Further, as the bone marrow failure aspects are soserious, there is need to provide new options to correct the associateddeficiencies. The identification and analysis of the gene that isaffected in SDS would provide for such opportunities.

Segregation analysis of an international collection of families of SDSpatients supports an autosomal recessive mode of inheritance (Ginzberget al., (2000), Am. J. Hum. Genet., v. 66, pp. 1413-1416). Previousstudies of families with SDS showed that the putative SDS locus mappedto the centromeric region of chromosome 7, to a 1.9 cM interval at7q11^(4,5). The genetic defect associated with the disease has, however,not previously been identified.

SUMMARY OF THE INVENTION

The invention provides a convenient and rapid method for the diagnosisof SDS, based on the finding that SDS is associated with mutations in apreviously uncharacterised gene residing within the 1.9 centiMorgandisease interval at 7q11 delineated by linkage and haplotype analysis infamily studies^(4,5). The gene, with a 1.6 kb transcript, was originallydesignated by the inventors as DEPCH and its encoded protein of 250amino acids was designated depechin. The gene has been renamed asShwachman-Bodian-Diamond Syndrome (SBDS) gene. A second copy previouslydesignated DEPCHP and now designated SBDSP, with 97% nucleotide sequenceidentity, resides within a locally duplicated genomic block of at least305 kb, and appears to be a pseudogene. Recurring mutations, theapparent result of recombination between the duplicated gene copies,were found in 89% of unrelated SDS patients (n=158), with 60% carryingtwo converted alleles and 29% having a different mutation in the secondallele. The extent of the converted segments varied but consistentlyincluded at least one of two critical sequence changes predicted toresult in truncation of the encoded protein. Other less common diseasealleles involve missense and insertion/deletion changes distinct fromthose in the pseudogene. The gene is a member of a highly conservedprotein family, with putative orthologues in diverse species rangingfrom archæbacteria to eukaryotes. The archaeal orthologues are locatedwithin highly conserved operons that include homologues of genesinvolved in RNA processing⁶, suggesting that SDS may be the result of adeficiency in some aspect of RNA metabolism that is essential forhæmatopoiesis, chondrogenesis and the development of the exocrinepancreas.

“SBDS or SBDS gene” is the chromosome 7q11.22 gene as described hereinwhich when mutated is associated with SDS. This definition includessequence polymorphisms wherein the nucleotide substitutions in the genesequence do not affect the function of the gene product.

“SBDS protein” is the protein encoded by the SBDS gene.

“Mutant SBDS gene” is the SBDS gene containing one or more mutationswhich, if present on both alleles of the gene, lead to SDS.

In accordance with one embodiment, the invention provides a method fordetermining whether a subject is suffering from Schwachman-DiamondSyndrome (SDS) comprising

obtaining a nucleic acid sample from the subject, and

conducting an assay on the nucleic acid sample to determine the presenceor absence of a SBDS gene mutation associated with SDS, wherein thepresence of a SBDS gene mutation associated with SDS in both SBDSalleles indicates that the subject suffers from SDS.

In accordance with a further embodiment, the invention provides a methodfor determining whether a subject is an SDS carrier comprising

obtaining a nucleic acid sample from the subject, and

conducting an assay on the nucleic acid sample to determine the presenceor absence of a SBDS gene mutation associated with SDS, wherein thepresence of a SBDS gene mutation associated with SDS in one SBDS alleleindicates that the subject is an SDS carrier.

In accordance with a further embodiment, the invention provides a methodfor determining whether a subject is suffering from Shwachman-DiamondSyndrome (SDS) comprising

obtaining a tissue sample from the subject, and

conducting an assay on the tissue sample to determine the level of SBDSprotein in the sample, wherein a reduced level of SBDS protein in thesample relative to a control sample indicates that the subject suffersfrom SDS.

In accordance with a further embodiment, the invention provides a methodfor determining whether a subject is at risk for developing acutemyelogenous leukaemia (AML) comprising

obtaining a nucleic acid sample from the subject, and

conducting an assay on the nucleic acid sample to determine the presenceor absence of a SBDS gene mutation associated with SDS, wherein thepresence of a SBDS gene mutation associated with SDS indicates that thesubject is at risk for development of AML.

In accordance with a further embodiment, the invention provides a methodfor treating a subject suffering from SDS comprising administering tothe subject a therapeutically effective amount of a substantiallypurified SBDS protein or of an isolated nucleotide sequence encoding anSBDS protein.

In accordance with a further embodiment, the invention provides anisolated nucleic acid molecule encoding an SBDS protein.

In accordance with a further embodiment, the invention provides anisolated nucleic acid molecule comprising at least about 10, 20, 30, 50,75 or 100 consecutive nucleotides of SEQ ID NO:1 or 29.

In accordance with a further embodiment, the invention provides asubstantially purified SBDS protein.

In accordance with a further embodiment, the invention provides anantibody which binds specifically to an epitope of an SDS protein.

In accordance with a further embodiment, the invention provides anucleotide sequence selected from the group consisting of:

(a) 5′-GCGTAAAAAGCCACAATAC-3′; (SEQ ID NO: 3) (b)5′-CTATGACAGTATTCGTAAGACTAGG-3′; (SEQ ID NO: 4) (c)5′-GGGGATTTGTTGTGTCTTG-3′; (SEQ ID NO: 5) (d)5′-CTTTCCTCCAGAAAAACAGC-3′; (SEQ ID NO: 6) (e)5′-AAATGGTAAGGCAAATACGG-3′; (SEQ ID NO: 7) (f)5′-ACCAAGTTCTTTATTATTAGAAGTGAC-3′; (SEQ ID NO: 8) (g)5′-GCTCAAACCATTACTTACATATTGA-3′; (SEQ ID NO: 9) (h)5′-CACTTGCTTCCATGCAGA-3′; (SEQ ID NO: 10) (i)5′-AAAGGGTCATTTTAACACTTC-3′; (SEQ ID NO: 11) (j)5′-GAAAATATCTGACGTTTACAACA-3′; (SEQ ID NO: 12) (k)5′-TCCACTGTAGATGTGAACTAACTC-3′; (SEQ ID NO: 13) (l)5′-CACTCTGGACTTTGCATCTT-3′; (SEQ ID NO: 14) (m)5′-GCTTCTGCTCCACCTGAC-3′; (SEQ ID NO: 15) (n)5′-AGCTATGCTGCAGCTGTTAC-3′; (SEQ ID NO: 16) (o)5′-ATGCATGTCCAAGTTTCAAG-3′; (SEQ ID NO: 17) (p)5′-TCCATGGCTATATTTTGATGA-3′; (SEQ ID NO: 18) (q)5′-TAAGCCTGCCAGACACAC-3′; (SEQ ID NO: 19) (r)5′-CACTCTGGACTTTGCATCTT-3′; (SEQ ID NO: 20) (s)5′-TGTTGGTTTTCACCGAATA-3′; (SEQ ID NO: 21) (t)5′-AGATAAAGAAAGACACACACAACT-3′; (SEQ ID NO: 22) (u)5′-GAAATCGCCTGCTACAAA-3′; (SEQ ID NO: 23) (v) 5′-TCAGCTTCTTGCCTTCAT-3′;(SEQ ID NO: 24) (w) 5′-TAAGTAAGCCTGCCAGACA-3′; (SEQ ID NO: 25) (x)5′-CATCAAGGTCTTTTTCCAAG-3′; (SEQ ID NO: 26) (y)5′-CCTGTCTCTGCCCAAGTC-3′; (SEQ ID NO: 27) and (z)5′-AGGGAACATTTTCAAAACTCA-3′. (SEQ ID NO: 28)

In accordance with a further embodiment, the invention provides atransgenic non-human mammal having within its genome an SBDS gene withat least one mutation associated with SDS.

In accordance with a further embodiment, the invention provides a kitcomprising at least one pair of primers suitable for amplification of atleast a portion of an SBDS gene.

SUMMARY OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows an integrated map of the interval of chromosome 7 where thegene deficiency that leads to SDS resides. a, The refined map interval,flanked by microsatellite markers D7S2429 and D7S502, is shown withreference to the Genbridge 3 radiation hybrid panel. b, An expanded mapof sub regions from RH bins 65 and 72 based on genomic sequences fromBAC clones in GenBank. The regions contains at least 305 kb that hasduplicated intrachromosomally. The positions and orientations of theparalogous duplicons along 7q were determined by unique STS content andradiation hybrid mapping. c, Identified genes in the BAC contigs areshown. Duplicon A contains at least 2 genes, SBDS and SDCR2A(Shwachman-Diamond Critical Region-2A). d, SBDS is composed of 5 exons(coding regions in grey, noncoding regions in black) spanning 7.9 kb ofgenomic sequence. The location of oligonucleotide primers used formutation screening by genomic PCR and RT-PCR are indicated.

FIG. 2 shows mutations in SBDS associated with SDS. a, Map of SBDS(coding regions in light blue) and sequence alignment of the exon 2region of SBDS and SBDSP, with gene-specific sequences in green andpseudogene sequences in red. In comparison to SBDS, SBDSP exon 2contains sequence changes (underlined in red) that are predicted toresult in truncation of its predicted protein product. These include anin-frame stop codon at 184 bp and a T>C change at 250+10 bp(corresponding to the invariant T of the donor splice site at 258+2 bpin SBDS) which results in the use of an alternate donor splice site(invariant splice site positions are boxed) at 250+1 bp. The sequencedifferences in SBDSP present restriction sites for Bsu36I, and DdeI at183 bp and Cac8I at 240+7 bp. b, Electropherograms for cloned sequencesfrom the exon 2 region of SBDS reveal sequence changes (red) derivedfrom gene conversion events between SBDS and its pseudogene; three geneconverted alleles are shown. These include [183TA>CT], [258+2T>C], andan extended conversion mutation [183TA>CT+201A>G+258+2T>C] with theintervening adenine (position 201) to guanine change. In each case,flanking sequences, including those at 129-2 bp and 258+124 bp, have notbeen converted (green). c, A restriction map of the SBDS exon 2 amplimer(primers E and F, FIG. 1 d) showing the position of Cac8I (C) and Bsu36I(B) restriction sites. Square brackets indicate the positions ofrestriction sites corresponding to converted sequences. The pedigree offamily SW20 is shown with affected individuals in black and carriers ingrey. Restriction fragment analysis of PCR amplified SBDS exon 2sequences revealed that the brothers inherited [183TA>CT] through thefather and paternal grandfather, and [258+2T>C] through the mother andmaternal grandmother. Patient P1 is heterozygous for [258+2T>C] and theextended conversion mutation ([183TA>CT+201A>G+258+2T>C]). Two unrelatedcontrol individuals are also shown (C1 and C2). d, Restriction maps ofthe gene and pseudogene loci showing the locations of all NdeIrestriction sites (N). Hybridisation of a DNA probe derived from apartial SBDS cDNA (green) to genomic DNAs restriction digested with NdeIindicates that members of family SW6 (including patient P1 with twoconverted alleles) show a pattern of hybridisation similar to twounrelated control individuals (C3 and C4) indicating that norearrangements or deletions have occurred in the vicinity of SBDS orSBDSP. e, Sequence traces depicting other representative codingmutations in patient SBDS compared to controls (N), including aninsertion ([96_(—)97insA]), a deletion ([119delG]) and two missensemutations ([24C>A] and [505C>T]).

FIG. 3 shows expression analysis of SBDS and SBDSP. FTh Fetal thymus,FSp Fetal spleen, FLi Fetal Liver, FK Fetal kidney, FSM Fetal skeletalmuscle, FLu Fetal lung, FH Fetal heart, FB Fetal brain, K Kidney, SMSkeletal muscle, Lu Lung, H Heart, B Brain, Li Liver, Pl Placenta, PaPancreas, Th Thymus, Sp Spleen, Ly Lymphocytes, To Tonsil, BM BoneMarrow, Le Peripheral Blood Leukocytes, LN Lymph Node, GAPDHGlyceraldehyde-3-Phosphate Dehydrogenase. a, RNA expression survey ofSBDS and SBDSP in primary tissues using a cloned RT-PCR productcontaining the entire SBDS open reading frame (primers T and R).Cumulative levels of both gene and pseudogene transcripts appear to belower in thymus and bone marrow. An alternatively spliced product wasdetected in several tissues and was most prominent in peripheral bloodleukocytes (Le). As shown in the lane indicated with an asterisk, thislarge transcript was detected with a probe derived from intron 1. b,Analysis of patient EBV-transformed B lymphoblastoid-derived RNA showsthat SBDS and SBDSP cumulative expression is lower in some patientscompared to a control individual (C). The probe used to provide acontrol for RNA loading consisted of a 983 bp cloned cDNA fragment fromglyceraldehyde 3-phosphate dehydrogenase (GAPDH). c, RT-PCR expressionanalysis of SBDS and SBDSP was carried out with specific oligonuleotideprimers and indicated that both transcripts are widely expressed.Sequencing of PCR products led to the identification of an exon2^(minus) transcript. RT-PCR indicated that the alternatively splicedproduct (shown as 349 bp) is present in all tissues tested, however itsexpression is significantly lower than transcripts that include exon 2(shown as 479 bp).

FIG. 4 shows CLUSTALX alignment of SBDS-encoded protein, SBDS, andrepresentative orthologues. Strong conservation is seen throughout thealignment from archæbacteria to complex eukaryotes. ‘*’ representsabsolutely conserved residues in the alignment, ‘:’ represents positionsat which conservative amino acid substitutions are observed and ‘.’represents semi conservative substitutions. The degree of sequencesimilarity is less pronounced towards the C-terminus although subgroupsretain strong conservation. The human amino acid sequence (Hsa) is shownin bold. The locations of all identified coding mutations arerepresented as white letters on a black background and correspondingamino acid sequence changes are shown above the alignment. A putativeU1-like zinc finger domain in three plant orthologues is indicated witha black bar. Ath Arabidopsis thaliana, Dme Drosophila melanogaster, CelCaenorhabditis elegans, Mmu Mus musculus, Hsa Homo sapiens, Ola Oryziaslatipes, Sce Saccharomyces cerevisiae, Ecu Encephalitozoon cuniculi, MacMethanosarcina acetivorans str. C2A, Hnr Halobacterium sp. NRC-1, MkaMethanopyrus kandleri str. AV19, Mja Methanococcus jannaschii, AfuArchaeoglobus fulgidus, Pab Pyrococcus abyssi, Tac Thermoplasmaacidophilum, Pae Pyrobaculum aerophilum, Sso Sulfolobus solfataricus,Ape Aeropyrum pernix, Pba Populus balsamifera, Gar Gossypium arboreum,⁺derived from partial GenBank EST sequence.

FIG. 5 shows the SBDS cDNA and its predicted encoded polypeptide. A: Thenucleotide sequence of the cDNA corresponding to SBDS mRNA is shownnumbered with the +1 starting at the first nucleotide, A, of thetranslation initiating codon. The 5′ and 3′ untranslated regions areshown in lower case, and the coding segment is shown in upper case text.B: amino acid sequence of the encoded polypeptide of 250 amino acids isshown numbered.

FIG. 6 shows the aligned genomic sequence for the human SBDS gene (SBDS)and its pseudogene SBDSP (SBDSP) and for the mouse SBDS gene (MUSBDS).The sequences for the five human exons are included with numbering thatcorresponds to that indicated in FIG. 5A. SBDS specific oligonucleotideprimers that can be used to determine the nucleotide sequence ofexpressed RNA or of each of the exons for mutation detection areindicated by underlining of the SBDS sequence. Dual specificoligonucleotide primers are indicated by the underlining of both SBDSand SBDSP sequences. The sequence of oligonucleotide primers indicatedin the forward direction (the arrows pointing to the right) corresponddirectly to the sequence shown, while those primers in the reversedirection (the arrows pointing to the left) are comprised of the reversecomplement of the indicated sequence.

FIG. 7 shows the specificity and reactivity of antibodies produced todetect the SBDS protein. a, Polyclonal antibodies produced withrecombinant SBDS (anti-rSBDS), left panel or a carboxyl peptide(anti-CpSBDS) of amino acids 224-239 (aa²²⁴IKKETKGKGSLEVLNL²³⁹ SEQ IDNO:29) of SBDS, right panel, detected single bands of the predicted sizein whole cell extracts of induced host E. coli BL21 containing thepET-28a expression vector with an in-frame fusion of the entire SBDSopen reading frame. A polyclonal antibody to an amino peptide(anti-NpSBDS) of amino acids 32-47 (aa³²CYKNKVVGWRSGVEKD⁴⁷ SEQ ID NO:30)of SBDS has also been generated, data not shown. b, The anti-rSBDSantibody also detected SBDS expressed transiently in HEK293 cells underthe control of a CMV promoter. The bands corresponds to those detectedby anti-Myc or anti-HA antibodies. The subtle shifts in sizes are due tothe various epitope tags and/or their locations that have been fused inframe to the SBDS gene, including amino or carboxyl positioned Myc(N-Myc or C-Myc) N-HA or amino or carboxyl positioned HA (N-HA or C-HA)tags. c, Anti-rSBDS also detected a prominent band in whole cellextracts of the predicted size for SBDS in BxPC3 (ATCC CRL-1687),SV40-transformed human fibroblasts (GM00639), Caco-2 (ATCC HTB-37),AR42J (ATCC CRL-1492), EBV transformed human lymphoblast (GM003798),PANC1 (ATCC CRL-1469) and J.RT3 (ATCC TIB-153) cell lines. The totalprotein loaded per extract is as indicated below each panel.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have identified the SBDS gene and described theassociation of mutations in that gene with the autosomal recessivedisease, SDS.

Clinical presentation in SDS can be variable but family studies havesupported a single gene locus near the centromere at 7q11^(2,4,5).Eighteen positional candidate genes were identified in compiled genomicsequences from the locus, and eight of these were analysed for mutationsin members of linked families. Disease-associated changes wereidentified in a gene represented by the full length, 1.6 kb cDNA cloneflj10917 (OVARC1000321). The gene was initially designated by theinventors as DEPCH (Development of Exocrine Pancreas, Chondrocytes andHæmatological lineages). The gene has been renamed (as approved by theHuman Genome Organisation Gene Nomenclature Committee) asShwachman-Bodian-Diamond Syndrome (SBDS) gene. The cDNA sequence isgiven in FIG. 5A (SEQ ID NO:1). SBDS is composed of 5 exons spanning 7.9kb, and is contained in BAC clone RP11-325K1. The nucleotide sequencesof the exons and surrounding introns are given in FIG. 6. The sequenceof murine SBDS is also shown in FIG. 6. SBDS and part of an adjacentgene reside in a block of genomic sequence of at least 305 kb that islocally duplicated (FIG. 1). The paralogous duplicon was mappeddistally, and contains an unprocessed pseudogene copy of SBDS, namedSBDSP. The pseudogene transcript is 97% identical to the SBDS transcriptwith small deletions and single nucleotide changes that clearly disruptcoding potential. The mouse and human SBDS genes have 88% nucleotideidentity and the proteins 97% amino acid identity, as seen in FIG. 6.

The protein product encoded by SBDS, termed SBDS, is a member of ahighly conserved protein family (Pfam UPF00023)²⁰. Orthologues exist inspecies ranging from archæbacteria to vertebrates and plants (FIG. 4).The sequence of 250 amino acids is given in FIG. 5B (SEQ ID NO:2) for apredicted polypeptide of 28.8 kDa with a pl of 8.9. The predicted aminoacid sequence has no homology to any known functional domain, and nosignal peptides were detected. The S. cerevisiae orthologue, encoded byORF YLR022c, has been found to bind specifically and with high affinityto the phospholipids Pl(4,5)P2 and Pl(4)P using yeast proteome chips²¹.The gene has also been deleted by the Yeast ORF Deletion Project andhaploid spores lacking YRL022c were found to be inviable²². Indirectlines of evidence suggest that orthologues of SBDS may play a role inRNA metabolism. First, YLR022c has been clustered with other genesencoding RNA processing enzymes based on microarray expression profileanalysis²³. In addition, SBDS archæl orthologues are located inconserved operons that contain several RNA processing genes, includinghomologues of subunits of the eukaryotic exosome and RNaseP complexes⁸.The A. thaliana orthologue, along with sequences derived from partialcDNAs from P. balsamifera and G. arboreum, have extended carboxyltermini corresponding to putative RNA-binding domains, suggesting afunctionally relevant fusion in flowering plants (FIG. 4). Theseobservations suggest that SDS may be the result of a defect in an RNAprocessing pathway. Manifestation of disease must reflect the loss orperturbation of a cellular function that is particularly critical forthe development of pancreatic acini, myeloid lineages, and chrondrocytesat growth plates of bones. The associated symptoms and the complicationsdue to bone marrow failure may reflect not only the loss of one gene butalso pleiotropic consequences of an aberrant pathway.

Sequence changes that do not alter protein-associated activities andthat occur in normal individuals are likely to correspond to genepolymorphisms. A current accepted standard to discriminate polymorphismsfrom mutations is to screen 100 individuals of comparable ethnicbackground that are not affected with SDS. Examples of polymorphismsdetected in SBDS are given in Table 2. SDS-associated mutations areshown in Table 1.

Diagnostic Methods

The invention provides a diagnostic method for determining whether asubject, such as a human subject, suffers from, or is at risk ofdeveloping, symptoms of SDS. In one embodiment, the method involvesexamining a nucleic acid sample from the subject for the presence orabsence of a mutation of the SBDS gene associated with SDS. Suchmutations include 183_(—)184TA→CT; 183_(—)184TA→CT+258+2T→C; 258+2T→C;24C→A; 96-97insA; 119 delG; 131A→G; 199A→G; 258+1G→C; 260T→G;291-293delTAAinsAGTTCAAGTATC; 377G→C; 505C→T; 56G→A; 93C→G; 97A→G;101A→T; 123delC; 279_(—)284delTCAACT; 296_(—)299delAAGA; 354A→C;428C→T+443A→G; 458A→G; 460-1G→A; 506G→C; and 624+1G→C. These mutationsare identified in relation to the numbering of the nucleotide sequenceof SEQ ID NO:1.

Many methods known to those of skill in the art can be used to detectthe presence or absence of a SBDS gene mutation in the subject's nucleicacid.

The cDNA sequence of the wild type SBDS gene is shown in FIG. 5 and isavailable at GenBank Accession Number AY169963 (NM_(—)016038). The exonstructure and flanking intron sequences are shown in FIG. 6.

“Mutations” of the wild type SBDS gene associated with SDS includeconversions, deletions, insertions, inversions or point mutations,either in the coding regions of the gene or gene regulatory regions.

A number of types of assay may be used to determine whether a subjecthas an SBDS gene mutation associated with SDS, including, for example,sequencing exons or other portions of the gene, including regulatory orintronic segments, PCR-RFLP analysis, allele specific PCR, allelespecific oligonucleotide hybridisation restriction fragment lengthpolymorphism (RFLP) analysis.

Where a direct sequencing assay is used, the sample may be DNA or RNA,for example genomic DNA or mRNA. Gene-controlling DNA segments and exonsof an individual can be amplified and then examined for direct sequencechanges, or scanned with methods that detect a heterozygous statefollowed by sequencing. These latter scanning methods can include singlestranded conformational analysis (Orita M, Iwahana H, Kanazawa H,Hayashi K and Sekiya T (1989), “Detections of polymorphisms of human DNAby gel electrophoresis as single-stranded conformation polymorphisms”,Proc. Natl. Acad. Sci, USA 86: 2776-2770), denaturing gradient gelelectrophoresis (Wartell R M, Hosseini S H and Moran C P Jr (1990),“Detecting base pair substitutions in DNA fragments bytemperature-gradient gel electrophoresis”, (Nucleic Acids Res. 18:2699-2705; Sheffield V C, Cox D R, Lerman L S and Myers R M (1989) or“Attachment of a 40-base-pair G+C rich sequence (GC clamp) to genomicDNA fragments by the polymerase chain reaction results in improveddetection of single-base changes” (Proc. Natl. Acad. Sci, USA 86:232-236); and denaturing high pressure liquid chromatography Cotton R GH, Edkins E, Forrest S (eds) 1998 “Mutation detection: a PracticalApproach” IRL Press, Oxford, and heteroduplex analysis Keen J, Lester D,Inglehearn C, Curtis A, Bhattacharya S (1991) Rapid detection of singlebase mismatches as heteroduplexes on Hydrolink gels. Trends Genet., 7:5,amongst other methods. Larger deletions or insertions can be detected bytraditional Southern blot analysis of DNA digest with restrictionenzymes (Southern E M. (1975) ‘Detection of specific sequences among DNAfragments separated by gel electrophoresis’, J Mol Biol 98:503-17).Mutant alleles can be distinguished by observing their inheritance fromeach parent and although each patient will have two affected alleles,they will typically appear in heterozygous state (all of the referencesof this paragraph are incorporated herein by reference).

The diagnostic methods of the invention are used to screen subjectsshowing symptoms of possible SDS, such as pancreatic insufficiency toidentify SDS, or to screen relatives of known SDS cases to determinewhether they may be at risk of developing SDS symptoms.

The diagnostic method of the invention should preferably be carried outon samples from children at a young age in order to establish thediagnosis and allow appropriate treatment. The diagnostic method mayalso be used as a prenatal test, using amniotic fluid or CVS samples.

With respect to determining carrier status, as discussed below, the testmay be carried out at any age, preferably at an age greater than 16years in relatives of SDS patients.

Signs of SDS generally are evident in children at an early age and thediagnostic methods of the invention will usually be employed todetermine if a child presenting with SDS symptoms is indeed sufferingfrom SDS. On occasion, a sibling or close relative may be screened todetermine if he or she suffers from SDS.

Suitable samples for testing of nucleic acid include buccal swabs, bloodsamples and bone marrow aspirates.

In one embodiment, genomic DNA is extracted from the sample and a targetportion of the genomic DNA comprising the SBDS gene or a selectedportion thereof is amplified by a polymerase chain reaction usingsuitable oligonucleotide primers, such as those described herein. Theamplified nucleic acid is then sequenced using conventional techniques.The sequence is compared with the wild type sequence to determine thepresence or absence of SDS-associated mutations. Primers must beselected which will amplify only the SBDS gene and not the pseudogene,as shown in FIG. 6. Since a larger number of SDS-associated mutationshave been observed in exon 2 of SBDS gene, it is preferable to lookfirst for mutations in that exon. If no mutations are found in exon 2,exons 1 and 3 to 5 are similarly examined in turn.

One of skill in the art can select suitable primers by reference to theSBDS sequence of FIG. 6, suitable primers are also identified inExample 1. Preferred primer pairs for amplification of SBDS exons are asfollows:

Exon 1: A & B or Q & B; Exon 2: E & F; Exon 3: G & H; Exon 4:SDCR9x4seqB; (5′-GCCTTCACTTTCTTCATAGT-3′: SEQ ID NO: 31) & J; and Exon5: SDCR9x5Fseq (5′-GCTTGCCTCAAAGGAAGTT-3′: SEQ ID NO: 32) & L.

Regulatory regions of SBDS, such as the promoter region, may also beexamined using suitable primers.

Promoter primers include SDCR9prom1RA (5′-CAGCCGACGACCTTGTTTT-3′: SEQ IDNO: 33) and SDCR9prom6FA (5′-GTGCCAACGCTGTGTTTT-3′: SEQ ID NO: 34).

These primers amplify a 501 bp segment partially overlapping exon 1,which likely contains the major controlling elements for thetranscription of SBDS mRNA.

For conversion mutations found in exon 2, examination of the testsubject's parents can be used to distinguish whether the subject has twoconversion recombinations rather than one extended conversionrecombination.

In a further embodiment of the invention, an RNA sample is obtained fromthe test subject and is reverse transcribed by conventional methods togive a corresponding cDNA which is amplified by PCR and sequenced.

In a further embodiment, RFLP analysis may be used to detect SBDS genemutations. Such methods of analysis are well known to those of skill inthe art and an example is described in the Examples herein and inreference 30. Test samples are compared with normal controls and samplesfrom patients with known mutations.

In a further embodiment, analysis of SBDS expression or of the level ofSBDS protein may used to determine whether a subject suffers from or isat risk of SDS. As described herein, SBDS is expressed in a wide varietyof tissues, including the most disease-relevant tissues, pancreas, bonemarrow and myeloid cell lineages. A blood or tissue sample may thereforebe used to evaluate SBDS expression or SBDS protein level. As seen inFIG. 3 b, mRNA level is notably reduced in SDS patients. SBDS expressioncan be evaluated by many routine methods, for example by mRNA analysisas described in the Examples herein and in reference 30.

In a further embodiment, an antibody specific for SBDS protein andcarrying a detectable label can be used to assess the level of SBDSprotein in a tissue sample of a subject by an immunological technique.Many suitable techniques, such as immunoprecipitation or ELISA assays,are known to those of skill in the art and are described, for example,in “Using antibodies—a laboratory manual”, (1999), Harlow et al., ColdSpring Harbor Lab. Press. The level of protein in a test subject iscompared with that in similar tissue samples from unaffectedindividuals, a reduction in level of SBDS protein being indicative ofSDS. The identification of the SBDS gene and the absence of any knownclosely related homologues enables the preparation of antibodies highlyspecific for SBDS protein.

Detection of SDS Carriers

The invention further provides a method for determining whether asubject is an SDS carrier by determining whether the subject has anSDS-associated mutation in one allele of the SBDS gene.

The methods described above for detecting an SDS-associated mutation ina sample from a subject suspected of suffering from SDS may also beapplied to detect carriers of the disease. The described methods fordetecting such mutations in a nucleic acid sample from a subject arepreferred.

Screening for SDS carriers is carried out especially on members offamilies with known SDS cases and may be important for geneticcounselling of such family members regarding their likelihood of passingthe disease on to their children. Generally, a method would be used tolook for a specific mutation already found in an affected family member.

Identification of Further Mutations

The present invention also enables the identification of additionalSDS-associated mutations of the SBDS gene, for example by examining SDSpatients using the methods and primers described herein.

Amplification of target portions of the gene, followed by direct nucleicacid sequencing, as described herein for diagnostic purposes, andcomparison with the wild type sequence, may be used to identifyadditional SDS-associated mutations.

Alternatively, assessment of the expression level of the SBDS gene, asdescribed herein, may indicate reduced expression levels and point tofurther mutations which can be characterised by nucleic acid analysis asdescribed above.

Nucleic Acids

The invention provides SBDS nucleic acids and homologues and portionsthereof. Preferred nucleic acids have a nucleotide sequence which is atleast 80%, preferably at least 90% and more preferably more than 97%homologous to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:29 orto a complement thereof.

Preferred nucleic acids are mammalian and especially preferred are humannucleic acids. Nucleic acids of the invention include nucleic acidsencoding an amino acid sequence with at least 75%, preferably at least90% and more preferably at least 99% amino acid identity to the aminoacid sequence of SEQ ID NO:2, and nucleic acids encoding a portion ofsuch amino acid sequences.

Also within the scope of the invention are nucleic acid molecules usefulas probes or primers and comprising at least about 10, 20, 30, 50, 75,90 or 100 consecutive nucleotides of SEQ ID NO:1.

Also within the scope of the invention are nucleic acids which hybridiseunder stringent conditions to a nucleic acid of the nucleotide sequenceSEQ ID NO:1 or to a complement or a portion thereof. Stringentconditions for nucleic acid hybridisation are known to those skilled inthe art and are described, for example, in “Protocols in MolecularBiology”, (1989), John Wiley & Sons, N.Y., at 6.3.1 to 6.3.6.

Also within the scope of the invention are nucleic acids which differfrom the sequence of SEQ ID NO:1 due to the degeneracy of the geneticcode.

Proteins

The invention provides substantially purified SBDS proteins and portionsthereof. These proteins and portions thereof are useful for thepreparation of antibodies specific for SBDS proteins.

“Substantially purified” as used herein with respect to proteins means aprotein preparation which is at least 75%, more preferably at least 90%and most preferably at least 99% by weight of SBDS protein.

Preferred SBDS proteins have an amino acid sequence which is at leastabout 75%, preferably at least about 90% and more preferably at leastabout 99% identical to the amino acid sequence of SEQ ID NO:2.

In a preferred embodiment, the SBDS protein has the amino acid sequenceof SEQ ID NO:2. Full length proteins and portions thereof correspondingto one or more domains thereof or comprising at least 5, 10, 25, 50, 75or 100 consecutive amino acids of SEQ ID NO:2 are within the scope ofthe invention.

The proteins and peptides of the invention may be isolated and purifiedby conventional protein purification methods such as gel-filtrationchromatography, ion exchange chromatography, high performance liquidchromatography, immunoprecipitation or immunoaffinity purification.

SBDS proteins may be prepared by conventional recombinant methods, forexample using the cDNAs described herein (for example human sequence hasGenbank Accession Number AY169963) or a selected portion thereof. Sincethe SBDS gene is small, native gene expression may be achieved with theincorporation of natural promoter and enhancer gene elements. Suitablevectors and host cells for such expression are well known to those ofskill in the art.

The expressed protein can be purified by standard procedures, asdescribed above.

Antibodies

The present invention also enables the preparation of antibodies orantibody fragments which bind specifically to SBDS protein or to aportion thereof.

The term “antibody” means a monoclonal antibody or a polyclonalantibody, which binds specifically to a particular peptide, polypeptideor epitope, i.e. with greater affinity than to other peptides,polypeptides or eptiopes, and includes chimeric antibodies, humanisedantibodies and single chain antibodies.

Chimeric antibodies are antibodies which contain portions of antibodiesfrom different species. For example, a chimeric antibody may have ahuman constant region and a variable region from another species.Chimeric antibodies may be produced by well known recombinant methods,as described in U.S. Pat. Nos. 5,354,847 and 5,500,362, and in thescientific literature (Couto et al., (1993), Hybridoma, 12:485-489).

Humanised antibodies are antibodies in which only the complementaritydetermining regions, which are responsible for antigen binding andspecificity, are from a non-human source, while substantially all of theremainder of the antibody molecule is human. Humanised antibodies andtheir preparation are also well known in the art—see, for example, U.S.Pat. Nos. 5,225,539; 5,585,089; 5,693,761 and 5,693,762.

Single chain antibodies are polypeptide sequences that are capable ofspecifically binding a peptide or epitope, where the single chainantibody is derived from either the light or heavy chain of a monoclonalor polyclonal antibody. Single chain antibodies include polypeptidesderived from humanised, chimeric or fully-human antibodies where thesingle chain antibody is derived from either the light or heavy chainthereof.

The term “antibody fragment” means a portion of an antibody thatdisplays the specific binding of the parent antibody and includes Fab, F(ab′)₂ and Fv fragments.

Polyclonal Antibodies

In order to prepare polyclonal antibodies, purified SBDS protein may beobtained, for example, as described herein. The purified protein or aportion thereof, coupled, if desired, to a carrier protein such asbovine serum albumin or keyhole limpet hemocyanin, as in Cruikshank W W,Center D M, Nisar N, Wu M, Natke B, Theodore A C, and Kornfeld H.,(1994), Proc. Natl. Acad. Sci. USA 24: 5109-5113, is mixed with Fruend'sadjuvant and injected into rabbits or other suitable laboratory animals.

Following booster injections at weekly intervals, the rabbits or otherlaboratory animals are then bled and the sera isolated. The sera can beused directly or purified prior to use by various methods includingaffinity chromatography employing Protein A-Sepharose, antigen Sepharoseor Anti-mouse-Ig-Sepharose. Further purification methods well known inthe art may be utilised to remove viral and/or endotoxin contaminants.

Monoclonal Antibodies

As will be understood by those skilled in the art, monoclonal antibodiesmay also be produced using an SBDS protein or a portion thereof. Theprotein or portion thereof, coupled to a carrier protein if desired, isinjected in Freund's adjuvant into mice. After being injected threetimes over a three-week period, the mice spleens are removed andresuspended in phosphate buffered saline (PBS). The spleen cells serveas a source of lymphocytes, some of which are producing antibody of theappropriate specificity. These are then fused with a permanently growingmyeloma partner cell, and the products of the fusion are plated into anumber of tissue culture wells in the presence of a selective agent suchas HAT. The wells are then screened by ELISA to identify thosecontaining cells making binding antibody. These are then plated andafter a period of growth, these wells are again screened to identifyantibody-producing cells. Several cloning procedures are carried outuntil over 90% of the wells contain single clones which are positive forantibody production. From this procedure a stable line of clones whichproduce the antibody is established. The monoclonal antibody can then bepurified by affinity chromatography using Protein A Sepharose,ion-exchange chromatography, as well as variations and combinations ofthese techniques. Truncated versions of monoclonal antibodies may alsobe produced by recombinant techniques in which plasmids are generatedwhich express the desired monoclonal antibody fragment in a suitablehost.

In a further embodiment, a cell line is provided which secretes anantibody specific for an SBDS protein or a portion thereof; a cell linesecreting an antibody specific for a human SBDS protein is preferred.

Diagnosis of Predisposition to AML

A number of SDS patients have been found to develop AML. It is of someconcern that individuals who have survived into adulthood without beingdiagnosed as SDS sufferers, because of minimal or unrecognised symptoms,may nevertheless also be at risk for the development of AML. The presentinvention permits the identification of these individuals as SDSsufferers, so that they may be monitored for early signs of AML andappropriately treated. Although widespread screening of the populationmay not be practical, screening of relatives of diagnosed SDS patientsfor SDS-associated mutations is completely feasible, as also would bescreening individuals exhibiting early or more overt signs of bonemarrow transformation.

In addition, SDS carriers, who have an SDS-associated mutation in onlyone allele of the SBDS gene and are therefore asymptomatic, may be atrisk for AML if they should experience loss or mutation of the wild-typeallele, particularly in haemotological tissues. Again, screening offamily members in SDS-affected families will indicate such geneticchanges.

Kits

The invention further provides kits for use in the diagnostic methodsdescribed above for determining whether a subject is suffering from oris at risk for SDS, for determining whether a subject is a carrier ofSDS or for determining whether a subject is at risk for AML. Such kitscan comprise, for example, one or more pairs of oligonucleotide primerssuitable for amplification of the SBDS gene or portions thereof, such asprimers suitable for amplification of particular exons of SBDS,particularly human SBDS, as described for example in FIG. 6. such kitscan also contain instructions for use of the primers, and optionally,additional reagents required for the diagnostic methods describedherein.

Therapeutic Methods

The invention further provides methods and compositions for treatingsubjects, including humans, suffering from SDS.

Methods of treatment are directed to restoring normal SBDS function inthe subject.

Such methods include gene therapy to restore normal function at the genelevel and administration of normal SBDS protein or portions thereof tomake up for lack of normal gene expression.

Gene therapy may, for example, involve administration to the subject ofa construct comprising an expression vector containing a nucleotidesequence encoding a wild type SBDS protein. Suitable expression vectorsinclude retroviral, adenoviral and vaccinia virus vectors.Administration may be intravenous, oral, subcutaneous, intramuscular orintraperitoneal.

A large number of gene delivery methods are well known to those of skillin the art and may include, for example liposome-based gene delivery(Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988)BioTechniques 6(7): 682-691; Rose U.S. Pat. No. 5,279,833; Brigham(1991) WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad Sci. USA84: 7413-7414), and replication-defective retroviral vectors harboring atherapeutic polynucleotide sequence as part of the retroviral genome(see, e.g., Miller et al. (1990) Mol. Cell. Biol. 10:4239 (1990);Kolberg (1992) J. NIH Res. 4:43, and Cornetta et al. Hum. Gene Ther.2:215 (1991)). Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmuno deficiency virus (SIV), human immuno deficiency virus (HIV), andcombinations thereof. See, e.g., Buchscher et al. (1992) J. Virol. 66(5)2731-2739; Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992);Sommerfelt et al., (1990) Virol. 176:58-59; Wilson et al. (1989) J.Virol. 63:2374-2378; Miller et al., J. Virol. 65:2220-2224 (1991);Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993) inFundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., NewYork and the references therein, and Yu et al., Gene Therapy (1994)supra).

AAV-based vectors are also used to transduce cells with target nucleicacids, e.g., in the in vitro production of nucleic acids and peptides,and in in vivo and ex vivo gene therapy procedures. See, West et al.(1987) Virology 160:38-47; Carter et al. (1989) U.S. Pat. No. 4,797,368;Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy5:793-801; Muzyczka (1994) J. Clin. Invest. 94:1351 and Samulski (supra)for an overview of MV vectors. Construction of recombinant MV vectorsare described in a number of publications, including Lebkowski, U.S.Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol.5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol. 4:2072-2081;Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol.63:03822-3828. Cell lines that can be transformed by rAAV include thosedescribed in Lebkowski et al. (1988) Mol. Cell. Biol. 8: 3988-3996.

The organ with the most serious life threatening consequences, the bonemarrow, may be treated by ex vivo gene therapy. This would involvethe 1) extraction of bone marrow cells, 2) introduction of cDNA withoutmutations in conjunction with expression guiding elements followed by 3)re-introduction of these modified cells back to the bone marrow. Similarstrategies have been used successfully in other diseases includingsevere combined immunodeficiency-X1 (M Cavazzana-Calvo, S Halcein-Bey, Gde Saint Basile, F Gross, E Yvon, P Nusbaum, F Selz, C Hue, S Certain,J-L Casanova, P Bousso, F Le Deist and A Fischer. (2000) Gene therapy ofhuman severe combined immunodefiency (SCID)-X1 disease. Science 288:669-672; all of which are incorporated herein by reference). The SBDSgene is notably small such that native gene expression may be achievedwith the incorporation of natural promoter and enhancer gene elements.

The SBDS nucleotide sequences described herein may be used inconventional expression systems, as described herein, to permitproduction of depechin protein in amounts sufficient for antibodyproduction or for therapy.

Therapeutic compositions in accordance with the invention comprise anisolated nucleotide sequence encoding an SBDS protein or effectivefragment thereof or a substantially purified SBDS protein or effectivefragment thereof.

Transgenic Animal Models of SDS

The invention further enables the creation of an animal model of SDSwhich is important for further study of how SBDS mutations lead to thevarious SDS-associated disease manifestations and for testing ofpotential therapeutics. A number of non-human mammals may be used tocreate such a model, including without limitation mice, rats, rabbits,sheep, goats and non-human primates. An animal model of SDS may havewithin its genome one or both SBDS genes with at least one mutationwhich when expressed results in symptoms of SDS. Identification andsequencing of the mouse SBDS gene homologue, as described herein,facilitates the creation of such animal models, for example a mousemodel.

Methods for the creation of transgenic animals are well known to thoseof skill in the art. A transgenic animal according to the invention isan animal having cells that contain a transgene which was introducedinto the animal or an ancestor of the animal at a prenatal (embryonic)stage. A transgenic animal can be created, for example, by introducingthe gene of interest into the male pronucleus of a fertilised oocyte by,e.g., microinjection, and allowing the oocyte to develop in apseudopregnant female foster animal. The gene of interest may includeappropriate promoter sequences, as well as intronic sequences andpolyadenylation signal sequences. Methods for producing transgenicanimals are disclosed in, e.g., U.S. Pat. Nos. 4,736,866 and 4,870,009and Hogan et al., A Laboratory Manual, Cold Spring Harbor Laboratory,1986. A transgenic founder animal can be used to breed additionalanimals carrying the transgene. A transgenic animal carrying onetransgene can also be bred to another transgenic animal carrying asecond transgene to create a “double transgenic” animal carrying twotransgenes. Alternatively, two transgenes can be co-microinjected toproduce a double transgenic animal. Animals carrying more than twotransgenes are also possible. Furthermore, heterozygous transgenicanimals, i.e., animals carrying one copy of a transgene, can be bred toa second animal heterozygous for the same transgene to producehomozygous animals carrying two copies of the transgene. For a review oftechniques that can be used to generate and assess transgenic animals,skilled artisans can consult Gordon (Intl. Rev. Cytol., 115:171-229(1989)), and may obtain additional guidance from, for example: Hogan etal, Manipulating the Mouse Embryo (Cold Spring Harbor Press, Cold SpringHarbor, N.Y. 1986); Krimpenfort et al., Bio/Technology, 9:844-847(1991); Palmiter et al., Cell, 41:343-345 (1985); Kraemer et al.,Genetic Manipulation of the Early Mammalian Embryo (Cold Spring HarborPress, Cold Spring Harbor, N.Y. 1985); Hammer et al., Nature,315:680-683 (1985); Purscel et al., Science, 244:1281-1288 (1986);Wagner et al., U.S. Pat. No. 5,175,385; and Krimpenfort et al., U.S.Pat. No. 5,175,384.

EXAMPLES

The examples are described for the purposes of illustration and are notintended to limit the scope of the invention.

Methods of molecular biology, genetics, protein and peptide biochemistryand immunology referred to but not explicitly described in thisdisclosure and examples are reported in the scientific literature andare well known to those skilled in the art.

Methods

Human Subjects. Families with SDS included in this study have beendescribed, and additional families have been obtained through ongoingrecruitment². The criterion for inclusion in the study was the presenceof both exocrine pancreatic dysfunction and hematologic abnormalities,including neutropenia and other problems associated with bone marrowfailure. Consent was obtained from all participating families, andprocedural approval was obtained from the human subjects review board ofThe Hospital for Sick Children, Toronto (HSC). Genomic DNA was extractedeither from Epstein-Barr virus (EBV) transformed B-lymphoblastoid celllines or directly from peripheral white blood cell pellets, as describedby Miller et al.²⁴. Patient and control RNA was extracted fromEBV-transformed B-lymphoblastoid cell lines as previously described²⁵.DNA from 100 control Caucasian individuals (Human variation panelHD100CAU) was purchased from Coriell Cell Repositories (Camden, N.J.).Physical Mapping. Genomic sequences were identified through BLASTanalysis of STSs and genetic markers in the SDS critical intervalagainst the GenBank non-redundant (nr) and high throughput genomesequence (htgs) databases²⁶. Where the density of pre-existing markerswas low, BAC and YAC clones assigned to the region were subcloned andsequenced to provide new STSs as described⁵. Genomic sequences werecompiled manually and the framework was supported by radiation hybridmapping of select STSs.Candidate Gene Identification. Candidate genes were identified ingenomic sequences through the use of annotation data provided by GenBank(http://www.ncbi.nlm.nih.gov) and Project Ensembl(http://www.ensembl.org)^(26,27). Ab initio gene predictions wereobtained through the use of GeneScript. Human genomic sequences werealso compared to mouse genomic sequences (available through CeleraDiscovery System and Celera Genomics' associated databases) from thesyntenic interval on mouse chromosome 5 using PipMaker2 to identifyregions of cross-species conservation²⁸. All in silico gene predictionswere confirmed by RT-PCR analysis using random-primed cDNA derived fromfetal brain, and/or testes poly(A)+ mRNA (Clontech, Palo Alto, Calif.).Mutation Detection. The genomic structure of the SBDS gene and itspseudogene copy were used to design primer pairs using Primer3 to screencoding regions²⁹. The position of primer pairs is shown (FIGS. 1 and 6).PCR products were directly sequenced or cloned using a Topo TA-cloningkit (Clontech) prior to sequencing. Primer pairs (specific for SBDSunless otherwise stated) used were: A (5′-GCGTAAAAAGCCACAATAC-3′: SEQ IDNO:3) and B (5′-CTATGACAGTATTCGTAAGACTAGG-3′: SEQ ID NO:4) (exon 1), C(5′-GGGGATTTGTTGTGTCTTG-3′: SEQ ID NO:5) and D(5′-CTTTCCTCCAGAAAAACAGC-3′: SEQ ID NO:6) (exon 2, SBDS/SBDSPdual-specific), E (5′-AAATGGTAAGGCAAATACGG-3′: SEQ ID NO:7) and F(5′-ACCAAGTTCTTTATTATTAGAAGTGAC-3′: SEQ ID NO:8) (exon 2), G(5′-GCTCAAACCATTACTTACATATTGA-3′: SEQ ID NO:9) and H(5′-CACTTGCTTCCATGCAGA-3′: SEQ ID NO:10) (exon 3), I(5′-AAAGGGTCATTTTAACACTTC-3′: SEQ ID NO:11) and J(5′-GAAAATATCTGACGTTTACAACA-3′: SEQ ID NO:12) (exon 4), K(5′-TCCACTGTAGATGTGAACTAACTC-3′: SEQ ID NO:13) and L(5′-CACTCTGGACTTTGCATCTT-3′: SEQ ID NO:14) (exon 5), M(5′-GCTTCTGCTCCACCTGAC-3′: SEQ ID NO:15) and N(5′AGCTATGCTGCAGCTGTTAC-3′: SEQ ID NO:16) (exons 1 & 2, SBDS/SBDSPdual-specific), O (5′-ATGCATGTCCAAGTTTCAAG-3′: SEQ ID NO:17) and P(5′-TCCATGGCTATATTTTGATGA-3′: SEQ ID NO:18) (exons 2 & 3, SBDS/SBDSPdual-specific). Patients were also screened for mutations throughsequencing of RT-PCR products from random-primed cDNA derived frompatient EBV-transformed B-lymphoblastoid cell lines. Primers used were:Q (5′-TAAGCCTGCCAGACACAC-3′ SEQ ID NO:19) and R(5′-CACTCTGGACTTTGCATCTT-3′ SEQ ID NO:20) (yields full length SBDS openreading frame), Q and S (5′-TGTTGGTTTTCACCGAATA-3′ SEQ ID NO:21), and T(5′-AGATAAAGAAAGACACACACAACT-3′ SEQ ID NO:22) and R. Gene conversionmutations were detected through restriction analysis of exon 2 PCRfragments. Exon 2 was amplified from patient DNA using PCR primers C & Dor E & F, and purified using a MinElute PCR Cleanup Kit (Qiagen).Restriction digestion using DdeI (not shown) or Bsu036I ([183TA>CT]) andCac8I ([258+2T>C]) (New England Biolabs, Beverly, Mass.) was carried outas recommended by the manufacturer and analyzed by agarose gelelectrophoresis. For all mutations, allele-specific oligonucleotidehybridisation to amplified SBDS exons from control individuals wascarried out as described³⁰.

The common mutations that account for the majority of SDS alleles can bedetected by PCR and restriction enzyme digestions by Bsu36I and Cac8I.These digestions can be performed singly or in combination.

PCR Amplification

Primer E: 5′-AAATGGTAAGGCAAATACGG-3′ (SEQ ID NO: 7) Primer F:5′-ACCAAGTTCTTTATTATTAGAAGTGAC-3′ (SEQ ID NO: 8)

-   -   product size: 733 bp; annealing temperature: 56.6° C.; extension        time: 40 sec        Double Digestion    -   Bsu36I (New England Biolabs #R0524): 6 units plus    -   Cac8I (New England Biolabs #R0579): 4.8 units per 100-200 ng PCR        product. Digest at 37° C.>3 hr        Band Sizes Detected on Agarose Gel with Ethidium Bromide        Intercalation    -   Normal: 584 bp also 64 bp, 41 bp, and smaller bands    -   258+2 T>C: 431 bp and 153 bp also 64 bp, 41 bp and smaller bands    -   183 TA>CT: 358 bp and 226 bp also 64 bp, 41 bp and smaller bands    -   258+2T>C+183TA>CT: 358 bp, 153 bp, 73 bp also 64 bp, 41 bp and        smaller bands        DdeI should not be used for this double digest; Bsu36I and Cac8I        should be used for this version of the assay.        Dual Specific Digests for Common Mutations        PCR Amplification

Forward Primer: 5′-GGGGATTTGTTGTGTCTT-3′ (SEQ ID NO: 5) Reverse Primer:5′-CTTTCCTCCAGAAAAACAGC-3′ (SEQ ID NO: 6)

-   -   product size: 336 bp; annealing temperature: 56° C.; extension        time: 1 min.        Cac8I Digest    -   Cac8I (NEB #R0579): 4.8 units; digest at 37° C.>3 hr        Band Size: Normal: 2×336 bp, 2×241 bp, 2×95 bp; 1 allele with        258+2 T>C: 1×336 bp, 3×241 bp, 3×95 bp; 2 alleles with 258+2        T>C: 4×241 bp, 4×95 bp.        Dde I Digest    -   Dde I (NEB #R0175): 6 units; digest at 37° C. 2 hr        Band Size: Normal: 2×190 bp, 2×169 bp, 4×146 bp, 2×21 bp; 1        allele with 183 TA>CT: 1×190 bp, 3×169 bp, 4×146 bp, 2×21 bp; 2        alleles with 183 TA>CT: 4×169 bp, 4×146 bp, 2×21 bp.        Southern Hybridisation. Genomic DNA from patients and control        individuals was subjected to restriction digestion with NdeI        (New England Biolabs) as recommended by the manufacturer and        products were separated by agarose gel electrophoresis. The DNA        was blotted and hybridised with a radiolabeled SBDS partial cDNA        probe (exons 1-3) as described³⁰.        RT-PCR and RNA Blot Analysis. A panel of cDNAs derived from 22        adult and fetal tissues (Clontech) were analyzed by RT-PCR        according the supplier's recommendations. Primers used were T        and R (SBDS), and (5′-TAAGTAAGCCTGCCAGACA-3′ SEQ ID NO:25) and        (5′-CATCAAGGTCTTTTTCCAAG-3′ SEQ ID NO:26) (SBDSP). Primers used        to assess the relative amount of SBDS exon 2 alternative        splicing were U (5′-GAAATCGCCTGCTACAAA-3′ SEQ ID NO:23) and V        (5′-TCAGCTTCTTGCCTTCAT-3′ SEQ ID NO:24). RNA blots of poly(A)+        mRNA (Clontech) were hybridized to DNA probes labeled with        [α³²P]-dCTP³⁰. The SBDS probe was a cloned RT-PCR fragment        (primers Q and R). The intron 1 probe was PCR amplified from        genomic DNA using primers (5′-CCTGTCTCTGCCCAAGTC-3′ SEQ ID        NO:27) and (5′-AGGGAACATTTTCAAAACTCA-3′ SEQ ID NO:28).        Sequence Alignment and Analysis. SBDS orthologues were        identified through BLASTP analysis of amino acid sequences in        the GenBank nr database, and through TBLASTN analysis of the        GenBank EST database (dbEST). Sequences were aligned with        CLUSTALX using default parameters followed by manual        adjustment³¹. Amino acids were analysed for the presence of        functional motifs using Pfam and associated databases        (http://www.sanger.ac.uk/Software/Pfam/)²¹.        Genbank Accession Numbers. SBDS consensus cDNA, AY169963 cDNA        flj10917, AK001779; SDCR2A (cDNA flj10900), AK001762; SDCR3        (cDNA flj10099), AK000961; BAC RP11-458F8, AC073335; BAC        RP11-325K1, AC079920; BAC RP11-584N20, AC069291; BAC        RP11-324F21, AC073089; BAC RP11-16604, AC006480; BAC        RP11-479C13, AC005236. Depechin orthologues: Arabidopsis        thaliana At1 g43860 gene product, NP_(—)564488; Drosophila        melanogaster CG8549 gene product, NP_(—)648057; Caenorhabditis        elegans protein W06E11.4.p, NP_(—)497226; Mus musculus protein        22A3, P70122; Oryzias latipes amino acid sequence derived from        cDNA clone MF01SSA157A09 5′ and 3′ overlapping sequence reads,        BJ013200 and BJ025159; Saccharomyces cerevisiae Ylr022cp,        NP_(—)013122; Encephalitozoon cuniculi ECU08_(—)1610 gene        product, NP_(—)597289; Methanosarcina acetivorans str. C2A        MA1778 gene product, NP_(—)616704; Halobacterium sp. NRC-1        Vng1276c, NP_(—)280149; Methanopyrus kandleri str. AV19 MK0384        gene product, NP_(—)613669; Methanococcus jannaschii MJ0592 gene        product, NP_(—)247572; Archaeoglobus fulgidus AF0491 gene        product, NP_(—)069327; Pyrococcus abyssi PAB0418 gene product        NP_(—)126299; Thermoplasma acidophilum Ta1291m gene product,        NP_(—)394745; Pyrobaculum aerophilum PAE2209 gene product,        NP_(—)559847; Sulfolobus solfataricus SSO0737 gene product,        NP_(—)342243; Aeropyrum pernix APE1167 gene product,        NP_(—)147753; Populus balsamifera subsp. Trichocarpa amino acid        sequence derived from cDNA clone F038P45Y, BI121507; Gossypium        arboreum amino acid sequence derived from cDNA clone        GA_Ed0050B07f, BQ402534.

Example 1

RT-PCR analysis of several SDS patients with SBDS-specificoligonucleotide primers (indicated as RT-PCR primers Q and R in FIG. 1 aand described in FIG. 6) revealed recurring sequence changes in exon 2,including a TA>CT dinucleotide change at position 183 or an 8 bpdeletion at the end of the exon (the nucleotide numbering is describedin FIGS. 5 and 6). Analysis of SBDS genomic sequences confirmed thepresence of the [183TA>CT] sequence change and revealed a [258+2T>C]nucleotide change in patients expressing the deleted SBDS transcript.[258+2T>C] is predicted to disrupt the donor splice site of intron 2,and the 8 bp deletion observed in the transcript is consistent with useof an upstream cryptic splice donor site at position 251. Alignment ofpatient SBDS sequences to genomic sequences from GenBank and controlindividuals indicated that both changes corresponded to sequencesnormally present in SBDSP (FIG. 2 a, b). The dinucleotide alteration[183TA>CT] introduces an in-frame stop codon (K62X) while [258+2T>C] andits resultant 8 bp deletion also causes premature truncation of theencoded protein by frameshift (84Cfs3). Patient alleles were alsoidentified that contain both of these changes together with anadditional silent nucleotide change ([201A>G]) in the interveningsegment, again consistent with the pseudogene sequence (FIG. 2 b). The[183TA>CT] and [258+2T>C] changes could be detected in amplified SBDSgenomic DNA followed by restriction digestion with Bsu36I and Cac8I,respectively (FIG. 2 a, c). Analysis of SDS pedigrees revealed thatthese changes were inherited and disease-associated. An example ofsegregating alleles in a linked pedigree is shown in FIG. 2 c. Thespecificity of genomic DNA amplimers for SBDS was supported by theabsence of additional pseudogene-like sequence changes in nucleotidepositions flanking the 183 and 258+2 bp positions (FIG. 2 b) and theabsence of any SBDSP-like sequences in 100 control samples. Thesefindings, together with the observation of unaltered hybridisationpatterns of genomic DNA with a SBDS probe (FIG. 2 d), indicated thatgene conversion due to recombination between SBDS and its highlyhomologous pseudogene had occurred. A similar basis for mutation hasbeen observed in other genetic diseases⁷⁻¹⁹. Sequence analysis of theexon 2 region of patients indicated that most conversion events areconfined to a short segment between 141 bp and 258+124 bp with a maximumsize of 240 bp (FIG. 2 a, b). Based on restriction digestion orsequencing of PCR products of patients from 158 unrelated families, 74%of SDS alleles (n=235 of 316) are the result of gene conversion, with89% of patients carrying at least one converted allele and 60% carryingtwo converted alleles. Consistent with being a recessive disease,patients carry mutations on both copies of the SBDS gene. Of thepatients analysed in the initial study, 50% were [183TA>CT]+[258+2T>C]compound heterozygotes, 5.1% were [183TA>CT+258+2T>C]+[258+2T>C]compound heterozygotes, and 4.4% were homozygous for a [258+2T>C]conversion. Of patient alleles not displaying the conversion mutations,genomic sequencing revealed other changes within the coding region ofSBDS, including small deletions, insertions, and nucleotidesubstitutions that would lead to frameshift and premature truncation,missense and nonsense changes (Table 1 and FIG. 4). To date, thesemutations were not detected in 100 Caucasian control DNA samples byallele specific oligonucleotide hybridization or correspond to changesof highly conserved amino acids that would not be expected to beimportant for protein structure or function. Table 1 shows theSDS-associated mutations identified in the initial study and insubsequent studies.

Example 2

RNA hybridisation with SBDS indicated broad expression of a 1.6 kbmessage (FIG. 3 a). Numerous GenBank EST clones, however, indicated thatthe pseudogene is also transcribed. Prominent larger-sized transcriptswere also observed in poly(A)+ mRNA from several tissues and wereconfirmed to include intron 1 through hybridisation of an intron1-specific probe (FIG. 3 a). In addition, three GenBank EST clonescorresponding to SBDSP were found to contain intron 1.

RNA expression analysis was carried out on a number of normal adult orfetal tissues, and on lymphoblasts from a number of SDS patients. Asseen from FIG. 3 b, the level of combined SBDS/SBDSP mRNA, andconsequently of protein product, was notably reduced in patient samples,compared with control C, lymphoblast RNA from a healthy subject.

Distinction between expression of the gene and pseudogene could beobtained through RT-PCR with specific oligonucleotide primers (FIG. 3c). Further, a broad survey of tissues revealed that the majority ofSBDS mRNA does contain exon 2 although its alternative splicing wasprominent in some patients (FIG. 3 c and data not shown). Both RT-PCRand RNA analyses supported widespread expression of SBDS in all tissuesexamined, including the most disease-relevant tissues, pancreas, bonemarrow, and myeloid lineages (FIG. 3 a, c).

Example 3

Generation of Antibodies for SBDS Protein Detection

Two methods were used to generate specific antibody probes to detectSBDS protein cells and tissues. First, a bacterially expressedpolypeptide with the entire open reading frame of SBDS and, second,specified peptides synthesised from the amino and carboxyl portion (seelegend to FIG. 7), were used as immunogens in rabbits. To obtain highlevel expression of recombinant SBDS, the complete open reading frame ofthe SBDS gene was incorporated into the pET28a vector (Novagen) usingstandard molecular biology techniques (Ref. 30). The open reading framewas fused with the (HIS)6 tag of the expression vector for purificationwith immobilised metal (Ni2+) affinity chromatography. The purifiedpolypeptide was then conjugated and injected into rabbits with theservices of Washington Biotechnology, Inc. Pre-immune and immune serawere collected and whole cell protein extracts of various cell typeswere assessed, FIG. 7. The amino and carboxyl peptide antibodies weresynthesised and prepared with the services of AnaSpec, Inc. andWashington Biotechnology, Inc., respectively. The antibodies showed highaffinity and specificity for the SBDS protein product in differentorgans and cell lines, by Western blotting carried out as follows.

Whole cell extracts were prepared with Laemmli (E. coli) or RIPA(mammalian cells) buffer (and separated by 13.5% PAGE prior to blottingon Hybond C Extra (Amersham) membrane (Ref. 30 and Harlow and Lane). ForrSBDS and anti-CpSBDS anti-sera, the membrane was blocked with 7% skimmilk in TBST (10 mM Tris HCl, pH7.3, 100 mM NaCl with 0.1% Tween 20) forovernight at room temperature followed by incubation of a 1:2000dilution for 5 h at room temperature. The blot was washed with TBST forfive consecutive washes and incubated with anti-rabbit secondaryantibody (Stressgen Biotechnologies Corp). The anti-Myc (OncogeneResearch Products) and anti-HA (BAbCO-Covance) monoclonal antibodies andthe anti-mouse secondary antibodies (Jackson ImmunoResearch Labs, Inc.)were used as recommended by their suppliers. The immunoreactive bandswere detected by enhanced chemiluminescence.

TABLE 1 SDS-associated mutations Predicted Amino Acid NucleotideSequence Changes Change 183_184TA→CT K62X 183_184TA→CT + 258 + 2T→C K62X258 + 2T→C 84Cfs3 24C→A N8K 96–97insA N34fs15 119delG S41fs17 131A→GE44G 199A→G K67E 258 + 1G→C 84Cfs3 260T→G I87S291–293delTAAinsAGTTCAAGTATC D97–K98delinsEVQVS 377G→C R126T 505C→TR169C 56G→A R19Q 93C→G C31W 97A→G K33E 101A→T N34I 123delC S41fs17279_284delTCAAGT Q94_V95del 296_299delAAAG E99fs20 354A→C K118N 428C→T +443A→G S143L + K148R 458A→G Q153R 460 − 1G→A splice 506G→T R169L 624 +1G→C splice

TABLE 2 SBDS Polymorphisms Some sequence changes in SBDS are predictedto be silent polymorphisms. Although some of these changes were detectedin SDS patients, allele-specific oligonucleotide hybridisation was usedto screen control samples to determine that these changes are notdisease associated and should be classified as silent polymorphisms.Nucleotide Sequence Change Predicted Amino Acid Change Intron 1 129 −71G→A 129 − 185G→A 129 − 225C→G 129 − 265G→A Intron 2 258 + 19A→G 258 +54T→G 258 + 99A→C Intron 3 459 + 92A→G Exon 2 141C→T L47L 201A→G K67KExon 5 651C→T F217F 635T→C I212T Rare Change 210T→C D70E

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1. A method for determining whether a subject is suffering fromShwachman-Diamond Syndrome (SDS) or is an SDS carrier comprisingobtaining a nucleic acid sample from the subject, and conducting anassay on the nucleic acid sample to determine the presence or absence ofa Shwachman-Bodian-Diamond-Syndrome (SBDS) gene mutation associated withSDS selected from the group consisting of 183TA>CT, 183TA>CT+258+2T>C,and 258+2T>C, and wherein the presence of said SBDS gene mutationassociated with SDS in both SBDS alleles indicates that the subjectsuffers from SDS and the presence of a SBDS gene mutation associatedwith SDS in one SBDS allele indicates that the subject is an SDScarrier.
 2. The method of claim 1 wherein the assay is selected from thegroup consisting of probe hybridisation, direct sequencing, restrictionenzyme fragment analysis and fragment electrophoretic mobility.
 3. Themethod of claim 2 wherein the nucleic acid sample is a DNA sample or anRNA sample and the assay is a direct sequencing assay.
 4. The method ofclaim 3 wherein the nucleic acid sample is a genomic DNA sample and theassay comprises the steps of: (a) amplifying a target portion of thenucleotide sequence of the genomic DNA; (b) obtaining the nucleotidesequence of said amplified target portion; and (c) determining thepresence or absence of said SBDS gene mutation associated with SDS insaid target portion of the nucleotide sequence.
 5. The method of claim 3wherein the nucleic acid sample is an RNA sample and the assay comprisesthe steps of: (a) reverse transcribing the RNA sample to produce acorresponding cDNA; (b) performing at least one polymerase chainreaction with suitable oligonucleotide primers to amplify the SBDS cDNA;(c) obtaining the nucleotide sequence of the amplified SBDS cDNA; and(d) determining the presence or absence of said SBDS gene mutationassociated with SDS in said nucleotide sequence.
 6. The method of claim4 wherein the target portion of the nucleotide sequence is amplifiedusing a primer pair selected from the group consisting of: (a)GCGTAAAAAGCCACAATAC and (SEQ ID NO: 3) CTATGACAGTATTCGTAAGACTAGG; (SEQID NO: 4) (b) AAATGGTAAGGCAAATACGG and (SEQ ID NO: 7)ACCAAGTTCTTTATTATTAGAAGTGAC; (SEQ ID NO: 8) (c)GCTCAAACCATTACTTACATATTGA and (SEQ ID NO: 9) CACTTGCTTCCATGCAGA; (SEQ IDNO: 10) (d) GCCTTCACTTTCTTCATAGT and (SEQ ID NO: 31)GAAAATATCTGACGTTTACAACA; (SEQ ID NO: 12) (e) GCTTGCCTCAAAGGAAGTT and(SEQ ID NO: 32) CACTCTGGACTTTGCATCTT; (SEQ ID NO: 14) (f)TAAGCCTGCCAGACACAC and (SEQ ID NO: 19) CTATGACAGTATTCGTAAGACTAGG; (SEQID NO: 4) (g) AAAGGGTCATTTTAACACTTC and (SEQ ID NO: 11)GAAAATATCTGACGTTTACAACA; (SEQ ID NO: 12) (h) TCCACTGTAGATGTGAACTAACTCand (SEQ ID NO: 13) CACTCTGGACTTTGCATCTT; (SEQ ID NO: 14) and (i)CAGCCGACGACCTTGTTTT and (SEQ ID NO: 33) GTGCCAACGCTGTGTTTT. (SEQ ID NO:34)


7. The method of claim 2 wherein the nucleic acid sample is a DNA sampleand the assay is a restriction enzyme fragment analysis.
 8. The methodof claim 7 wherein the assay comprises the steps of: (a) digesting theDNA with a restriction enzyme to give restriction fragments; (b)separating the restriction fragments by agarose gel electrophoresis; and(c) detecting the separated fragments by hybridisation of the fragmentsto a detectably labelled nucleotide probe specific for SBDS.
 9. Themethod of claim 8, wherein the method is for determining whether asubject is suffering from SDS and wherein the restriction enzyme is atleast one of Cac81 and Bsu361.
 10. The method of any one of claims 1 to9 wherein the subject is a human subject.
 11. The method of claim 8,wherein the method is for determining whether a subject is an SDScarrier and wherein the restriction enzyme is Nde 1.